Abstract
Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.
Highlights
Clinical trials with Chimeric Antigen Receptor (CAR)-T cells redirected to target the pan-B cell antigen CD19 have shown promising results in treating children and adults with B-cell acute lymphoblastic leukemia [1, 2]
We presented a systematic method to study activation and degranulation in CAR-T cells using antigen-presenting cell surfaces
The level of activation of non-transduced T cells (NT) activating through the TCR and CD19.CAR-T cells activating through the CAR
Summary
Clinical trials with CAR-T cells redirected to target the pan-B cell antigen CD19 have shown promising results in treating children and adults with B-cell acute lymphoblastic leukemia [1, 2]. Activation and degranulation of CAR-T cells performed in part at the Joint School of Nanoscience and Nanoengineering, a member of the Southeastern Nanotechnology Infrastructure Corridor (SENIC) and National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant ECCS-1542174). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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